Gel‐like inclusions of C‐terminal fragments of TDP‐43 sequester stalled proteasomes in neurons

Abstract Aggregation of the multifunctional RNA‐binding protein TDP‐43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C‐terminal fragments of ~25 kDa ("TDP‐25") accumulate in cytoplasmic inclusions. Here, we analyze gain‐of‐function mechanisms of TDP‐25 combining cryo‐electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP‐25 inclusions are amorphous, and photobleaching experiments reveal gel‐like biophysical properties that are less dynamic than nuclear TDP‐43. Compared with full‐length TDP‐43, the TDP‐25 interactome is depleted of low‐complexity domain proteins. TDP‐25 inclusions are enriched in 26S proteasomes adopting exclusively substrate‐processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP‐25 impairs proteostasis, and this inhibitory function is enhanced by ALS‐causing TDP‐43 mutations. These findings support a patho‐physiological relevance of proteasome dysfunction in ALS/FTD.


12th Oct 2021 1st Editorial Decision
Dear Dieter, Thank you for the submission of your manuscript to EMBO reports. We have now received the full set of referee reports that is pasted below.
As you will see, the referees acknowledge that the findings are potentially interesting. However, they also raise several concerns, and I think all should be addressed. Especially the physiological relevance of the TDP-25 peptide should to be strengthened. If you disagree or have any other comments, we can also discuss the revisions in a video chat, if you like.
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3) We replaced Supplementary Information with Expanded View (EV) Figures and
-Please also include scale bars in all microscopy images.
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Best wishes, Esther
Esther Schnapp, PhD Senior Editor EMBO reports Referee #1: In this work, Riemenschneider have investigated the type of inclusions formed by a toxic C-terminal fragment of TDP-43 called TDP-25. Until this moment, the toxic mechanisms promoted by this fragment have never been clarified. In this work, the authors have combined several state-of-the-art techniques to provide an explanation. From their studies, the authors have concluded that TDP-25 toxicity could principally stems from an impairment of the cellular proteasomal machinery. In general, the experiments have been performed to very high technical standards and results are convincing. A few additions/clarifications would nonetheless help to strengthen the conclusions of the manuscript: -The data in Figure 3F suggest that at high expression levels, TDP-25 mut impaired overall protein degradation significantly more than TDP-25 wt. Looking at the curves in this figure, this certainly seems to be the case. However, no significance values are reported in the inset with regards to tagRFP-TDP-25 wt vs. tagRFP-TDP-25 mut, they are only referred to tagRFP. Have these values been calculated?. In this experiment, it would have been interesting to see how these fragments compare with the TDP-43 wt protein.
-Most interestingly, the considerable difference between TDP-25wt and TDP-25mut in Figure 2D, compared to the high similarity between these two fragments in the structural and photobleaching experiments, would suggest that the explanation may reside in the protein interaction profile. Have the authors considered performing a proteomic experiment with GFP-TDP-25 mut and compared the profile with the one obtained using GFP-TDP-25 wt? -Related to this, one of the most striking data from this study is that compared to TDP-43 wt the TDP-25 fragment loses only 72 interactions but gains more than 400 ones. However, the TDP-25 sequence is completely present in the TDP-43 wt sequence and this raises the question how this can occur. This aspect is not discussed in the manuscript and, although additional experiments may be beyond the scope of this work, some speculation could be added to improve Conclusions. For example, could the structures in Fig.1E be particularly "sticky" for proteasome components?.
-Finally, one important aspect that has not been addressed in this study is the potential physiological importance of this TDP-25 mediated proteasomal inhibition in physiological conditions. It must be noted, in fact, that the presence of the 25kDa fragment in patient neuronal tissue does not seem to occur at the very high levels used in this study. Indeed, in all western blots from ALS/FTLD-TDP patients the presence of the 25kDa (and sometimes 35kDa) fragments does not seem to appreciably reduce the amount of full length TDP-43 present in the sample. This raises the question whether the proteosomal inhibition mediated by TDP-25 could represent a pathological effect even when present in lesser quantities. At the very least, this is a caveat that should be discussed in Conclusions. Ideally, the authors could see whether the structures identified in the CryoEM experiments or increased proteasomal inhibition also exist in cells where 25 kDa production can be induced to somewhat similar pathological levels using various chemicals (ie. Dexamethasone, L-BMAA, para-Chloroamphetamine).

Referee #2:
This is a well-written paper to use three pronged approaches of cryo-ET, mass spec and functional assays to tackle a complex neurodegeneration problem on where and how the TDP-25 impact on cellular functions. The conclusion that proteasome dysfunction is a hallmark of ALS/FTD pathogenesis is not surprising and opens more avenues in therapeutic strategies. However, I have a few technical questions on the cryoET analysis that needs to be clarified in the text or the supplement.
1. How do they pick the aggregates (i.e., the annotated red features) in Figure 1D to be GFP-TDP-25? They are variable sizes and shapes. Was a control done from cells without GFP-TDP-25? 2. Are the TDP-25 inclusions localized only in certain region of the cells?
3. Is there any cryo-ET structural difference between the wildtype and mutant GFP-TDP-25 inclusions? 4. I am concerned about using template to pick ribosomes, proteosomes and TRiC. Have they carried out independent approach to validate the picked proteosomes to be in the same structural state? Again, a side by side control experiment without TDP-25 present cell would be great.
5. What are the particle statistics to derive the plot in Supp Figure 1C? 6. What are the cryoET data statistics before the conclusions are made. How do the plots shown in Supplement Figure 1D compared between cells without and with TDP-25 transduction.
7. It is necessary that at least one representative tomogram reconstruction is deposited to the EMDB for the public use. In addition, the subtomogram average of the proteasome along with the FSC plot should also be deposited.
Referee #3: The manuscript by Riemenschneider et al. describes the characterisation of aggregation of a truncated version of TDP-25 in to inclusions. The paper reports the formation of solidified structures that appear to be non-fibrillar and associate with stalled conformation of proteosomes. The paper is well written but the shortened format has lead to some questions regarding the approaches that are a unclear to the reader. The paper includes a large amount of electron tomography but the description of it really enough lacks detail. There is a lack of information on repeats, statistics and analysis methods. Figure 1A shows a single example of co-localisation of TDP25 (wt or mutant) with P-TDP43. There is no quantification or statistics here. Figure 1B shows a single WB again with no quantification or stats. In Figure 1C, how have TDP25 containing inclusions been identified in the cells? It would be helpful to include further explanation of the annotation process for ET. These additional details are necessary to give confidence for the reported results.
Minor : 1. the abstract states that TDP43 correlates with FTD but in the introduction, it is more correctly stated that it correlates with 45% of FTD, so the abstract should be amended (some cases of FTD).
2. This sentence in the introduction needs attention: "Here, we aimed to elucidate gain-of-function mechanisms as well as the structure of cytoplasmic TDP-43 aggregates found in sporadic and most genetic ALS/FTD cases we focused on the aggregation-prone TDP-25 fragment (residues 220-414 of full length human TDP-43) (Neumann et al, 2006; Zhang et al, 2009) using a pipeline of cryo-ET, proteomics and functional assays" Overall, this is an interesting paper but I feel unsure of the use and significance of the truncation version of TDP43. The authors may need to defend their use of this fragment further for the physiological relevance of their findings for ALS and FTD that involves TDP43 -The data in Figure 3F suggest that at high expression levels, TDP-25 mut impaired overall protein degradation significantly more than TDP-25 wt. Looking at the curves in this figure, this certainly seems to be the case. However, no significance values are reported in the inset with regards to tagRFP-TDP-25 wt vs. tagRFP-TDP-25 mut, they are only referred to tagRFP. Have these values been calculated? In this experiment, it would have been interesting to see how these fragments compare with the TDP-43 wt protein.

In this work, Riemenschneider have investigated the type of inclusions formed by a toxic C-terminal
We thank the reviewer for this comment. We added the wild-type and mutant full-length protein (tagRFP-TDP-43) in the revised Figure 3F and provide a detailed statistical analysis in the new Fig.  EV3C to maintain clarity with the higher number of comparisons. While low levels of RFP-TDP-43 have minimal effect compared to the RFP control, high level expression inhibit proteasomal processing of the reporter construct, which is consistent with proteasomal enrichment also in the TDP-43 full length interactome (see Figure 3A), albeit to a lesser extent than in the TDP-25 interactome. Interestingly, a full length TDP-43 containing the eight ALS-causing mutations further increased the reporter levels at high expression levels, without reaching statistical significance.
-Most interestingly, the considerable difference between TDP-25wt and TDP-25mut in Figure 2D, compared to the high similarity between these two fragments in the structural and photobleaching experiments, would suggest that the explanation may reside in the protein interaction profile. Have the authors considered performing a proteomic experiment with GFP-TDP-25 mut and compared the profile with the one obtained using GFP-TDP-25 wt?
We assume this comment refers to Figure 3F. We followed this excellent suggestion and compared the interactomes of wild-type and mutant TDP-25 (extended Figure EV3D and Table S3). In these replicate experiments the interactome of wild-type TDP-25 was strongly enriched in proteasomal subunits, which convincingly reproduces our initial findings. Furthermore, the interaction profile of TDP-25 mutant also revealed a prominent enrichment of proteasomal subunits.
While the overall interaction profiles were comparable, mutant TDP-25 interacted weaker with Hspa1a, a member of the Hsp70 family, and Bag2, a Hsp70/Hsc70 interacting co-chaperone, but stronger with the proteasome core subunit Psma4 (new Figure EV3D). Thus, the effects of the ALS mutations may be indirect, e.g. through proteins co-partitioning into the inclusions without direct interaction with TDP-25. Moreover, introducing the same mutations into full length TDP-43 also impaired proteostasis more than wild-type TDP-43 (revised Fig 3F).
-Related to this, one of the most striking data from this study is that compared to TDP-43 wt the TDP-25 fragment loses only 72 interactions but gains more than 400 ones. However, the TDP-25 sequence is completely present in the TDP-43 wt sequence and this raises the question how this can occur. This aspect is not discussed in the manuscript and, although additional experiments may be 17th Jan 2022 1st Authors' Response to Reviewers beyond the scope of this work, some speculation could be added to improve Conclusions. For example, could the structures in Fig.1E be particularly "sticky" for proteasome components?
It is indeed counterintuitive that a protein fragment has more interactors than the full-length protein. However, this effect could be explained by co-partitioning of other proteins into the gel-like inclusions. Moreover, protein misfolding rapidly recruits the UPS machinery and other proteins that are also ubiquitinated (e.g. Gottlieb et al, JBC 2019). Finally, our data is consistent with findings from Chou et al (Nat Neuro 2017) using proximity labeling.
The extra density in Fig 1E likely reflects substrates or adaptor proteins as mentioned in the result section. We do not have structures of proteasomes in TDP-43 overexpressing cells and thus cannot tell whether this is TDP-25 specific material. However, we noticed similar extra densities in poly-GA expressing cells (Guo et al, Cell 2018). Although the resolution of the extra density does not allow its molecular identification, it is localized to a specific region of the proteasome, suggesting that the interaction is specific at least to some extent.
-Finally, one important aspect that has not been addressed in this study is the potential physiological importance of this TDP-25 mediated proteasomal inhibition in physiological conditions. It must be noted, in fact, that the presence of the 25kDa fragment in patient neuronal tissue does not seem to occur at the very high levels used in this study. Indeed, in all western blots from ALS/FTLD-TDP patients the presence of the 25kDa (and sometimes 35kDa) fragments does not seem to appreciably reduce the amount of full length TDP-43 present in the sample. This raises the question whether the proteosomal inhibition mediated by TDP-25 could represent a pathological effect even when present in lesser quantities. At the very least, this is a caveat that should be discussed in Conclusions. Ideally, the authors could see whether the structures identified in the CryoEM experiments or increased proteasomal inhibition also exist in cells where 25 kDa production can be induced to somewhat similar pathological levels using various chemicals (ie. Dexamethasone, L-BMAA, para-Chloroamphetamine).
We thank the reviewer for this comment and acknowledge the value of the suggested experiment. The research community still lacks a sophisticated model system that shows C-terminal fragmentation without relying on overexpression of TDP-43 species. Despite our efforts, we couldn't detect any C-terminal TDP-43 fragments upon treatment with various concentrations of Dexamethasone (= Dexam., toxic at 500µM), L-BMAA, BAPTA-AM (toxic at 100µM), EDTA (toxic at 2.5-100mM) or 15d-PGJ2 (toxic at 20µM) for up to 24h in the UbG76V-GFP reporter HEK293 cell line (see Figure below). The ordered para-Chloroamphetamine was never delivered and could not be tested. Therefore, we unfortunately could not assess the effect of "endogenous" TDP-25 levels on proteasome activity. Moreover, we didn't test compounds in primary neurons, because they would presumably induce very small aggregates (similar to MG132 treatment, Khosravi et al, EMBO J 2020), which are beyond the CLEM resolution and would not allow analysis by cryo-ET.
However, comparison with RFP and the newly added RFP-TDP-43 clearly shows that RFP-TDP-25 increases reporter levels in all but the two lowest intensity bins, which strongly argues for proteasome inhibition also at low expression levels, although the difference between wild-type and mutant TDP-25 only becomes apparent at high expression levels (revised Fig 3F and new EV3C). We emphasized this point in the revised manuscript.
Western Blot analysis of UbG76V-GFP reporter cell line treated with different compounds reported to induce TDP-43 C-terminal fragmentation. UbG76V-GFP HEK293 cells were treated with the indicated compound or the respective vehicle control (DMSO for Dexamethasone and BAPTA-AM, NaHCO3 for L-BMAA or H2O for EDTA) for 24h. Total cell lysates were immunoblotted for TDP-43 using an anti-TDP-43 C-terminal antibody (proteintech #12892-1-AP). β-Actin served as a loading control.

This is a well-written paper to use three pronged approaches of cryo-ET, mass spec and functional assays to tackle a complex neurodegeneration problem on where and how the TDP-25 impact on cellular functions. The conclusion that proteasome dysfunction is a hallmark of ALS/FTD pathogenesis is not surprising and opens more avenues in therapeutic strategies. However, I have a few technical
questions on the cryoET analysis that needs to be clarified in the text or the supplement.
1. How do they pick the aggregates (i.e., the annotated red features) in Figure 1D to be GFP-TDP-25? They are variable sizes and shapes. Was a control done from cells without GFP-TDP-25?
We apologize for the brief description in the original manuscript. We added a small section describing the procedure in the result section and extended the legend for Fig. 1D in the revised manuscript. In general, the identification is based on correlative light-electron microscopy (CLEM). Cryo-FIB lamella preparation was always guided by the cryo-LM information: the lamella was only prepared in the region with GFP signal (additional detailed information can be found in the Methods section).
We now indicate in the legend of Fig. 1 that the irregular aggregate structures were approximately segmented using a threshold-based approach for visualization purposes. The perimeter of the aggregate was approximately defined visually as the contour of the amorphous aggregate.
Reaching sufficient resolution to determine proteasome conformation requires a high number of particles, which can be much more easily achieved in conditions where proteasomes are concentrated such as TDP-25 inclusions. Analyzing a comparable number of particles in control cells would require an enormous amount of data, and thus we refer to a previous study where such a dataset was collected (Asano et al. 2015).

Are the TDP-25 inclusions localized only in certain region of the cells?
We found the TDP-25 inclusions to be mainly localized in the neuronal soma. This is now indicated in the revised manuscript. Occasionally, smaller inclusions could be identified in dendrites, but they were too small to process in our CLEM/cET pipeline.

Is there any cryo-ET structural difference between the wildtype and mutant GFP-TDP-25 inclusions?
This is an interesting question. With the current resolution we could not detect any differences between wild-type and mutant GFP-TDP-25 inclusion as indicated in the revised manuscript.

I am concerned about using template to pick ribosomes, proteosomes and TRiC. Have they carried out independent approach to validate the picked proteosomes to be in the same structural state? Again, a side by side control experiment without TDP-25 present cell would be great.
This is a valid remark. We applied the same approach of template matching and proteasome averaging as in our previous work (Guo et al, Cell, 2018). In that study, we showed that our computational workflow can distinguish different proteasome conformations if they are present. In brief, to avoid the influence of the template induced bias, low-resolution (~40 Å in resolution) templates were always used. Thus, the higher-resolution details visible in averages must originate from the data itself and not from reference bias. This process is illustrated below for ribosomes within our tomograms, showing the template (left) and averaging result (right). Proteasome averages also gained higher-resolution features, as the ground and substrate processing conformations could not be distinguished at the resolution of the template. Additional features not present in the template were also observed in the average, such as the extra densities shown in Fig.  1E.
For the structural states analysis, all the subtomograms were used for multiple round of classification using RELION, which is a regularized likelihood based classification approach. The method is widely used in the field of cryo-EM/ET to separate conformationally heterogeneous particles. However, with this approach small subgroups with less than 10% of the total populations are difficult to separate. We have now added a sentence to the text acknowledging that a small fraction of proteasome particles in other conformations may be present. Figure 1C?

What are the particle statistics to derive the plot in Supp
In total 1723 proteasome caps were used for the final refinement as indicated in the revised legend. For the resolution estimation, we performed the gold-standard FSC calculation (Scheres, S. H, Nat. Methods, 2012): the subtomograms were separated randomly to two halves and perform refinement independently to get the two volumes for FSC calculation.  Note, that proteasome concentration was calculated for the whole tomographic volume, not only in the area occupied by the inclusion 7. It is necessary that at least one representative tomogram reconstruction is deposited to the EMDB for the public use. In addition, the subtomogram average of the proteasome along with the FSC plot should also be deposited.
We deposited the tomographic reconstruction shown in Fig 1C at EMDB with the accession code EMD-32217.
The subtomogram average of 26S proteasomes within neurons with TDP-25 inclusions has also been deposited at EMDB with the accession code EMD-32216.

The manuscript by Riemenschneider et al. describes the characterisation of aggregation of a truncated version of TDP-25 in to inclusions. The paper reports the formation of solidified structures that appear to be non-fibrillar and associate with stalled conformation of proteosomes.
The paper is well written but the shortened format has lead to some questions regarding the approaches that are a unclear to the reader. The paper includes a large amount of electron tomography but the description of it really enough lacks detail. There is a lack of information on repeats, statistics and analysis methods.
We thank the reviewer for the interest in our study and the constructive comments. We expanded the description of the cryo-electron tomography section to make it easier comprehensible, but unfortunately, we are restricted by a tight word count. Moreover, we provide detailed statistical information for figures 2B and EV2. Figure 1A shows a single example of co-localisation of TDP25 (wt or mutant) with P-TDP43. There is no quantification or statistics here. Figure 1B shows a single WB again with no quantification or stats.
Quantification of the western blots shows that solubility of wild-type and mutant TDP-25 is similar (new Fig 1C).
In Figure 1C, how have TDP25 containing inclusions been identified in the cells? It would be helpful to include further explanation of the annotation process for ET. These additional details are necessary to give confidence for the reported results.
We added a small section describing the procedure in the result section and extended the legend for Fig. 1D in the revised manuscript. In general, the identification is based on correlative light-electron microscopy (CLEM). Cryo-FIB lamella preparation was always guided by the cryo-LM information: the lamella was only prepared in the region with GFP signal (additional detailed information can be found in the Methods section).
We For the annotation, the inclusions were segmented by low-pass filtering and thresholding the higher intensity regions in Amira. The membranes were segmented with TomoSegMemTV software, with the missing region fixed manually in Amira. Molecules like proteasomes, ribosomes, TRiC were identified by template matching and pasted back with the position and Euler angles determined by template matching results (refinement star file in the case of proteasome). Additional information on these procedures has been included in the revised text. Minor: 1. the abstract states that TDP43 correlates with FTD but in the introduction, it is more correctly stated that it correlates with 45% of FTD, so the abstract should be amended (some cases of FTD).
We adjusted the abstract in the revised manuscript accordingly.

This sentence in the introduction needs attention:
"Here, we aimed to elucidate gain-of-function mechanisms as well as the structure of cytoplasmic TDP-43 aggregates found in sporadic and most genetic ALS/FTD cases we focused on the aggregation-prone TDP-25 fragment (residues 220-414 of full length human TDP-43) (Neumann et al, 2006;Zhang et al, 2009) using a pipeline of cryo-ET, proteomics and functional assays" We correct our grammar mistake.
Overall, this is an interesting paper but I feel . Surprisingly, high expression levels of TDP-43 inhibit proteostasis (which is consistent with less pronounced proteasome recruitment revealed by mass spectrometry, see Fig 3A) in the absence of inclusion formation, and introducing eight ALS-causing mutations has an even bigger effect suggesting that our findings also have implications for full-length TDP-43.
14th Mar 2022 1st Revision -Editorial Decision Dear Dieter, Thank you for the submission of your revised manuscript. We have now received the enclosed reports from the referees that were asked to assess it, and I am happy to say that both referees support the publication of your study now. We can therefore in principle accept it.
Only a few editorial requests still need to be addressed: -Please upload all main and EV figures as separate, individual files.
-The Fig EV3A callout is missing, please add.
-Please upload the movie files as individual movies together with their legends in zipped files.
-Please make sure that all data deposited in public repositories are freely accessible to the readers upon the publication of this paper.
-The EV figure legends should be moved to after the main figure legends. Please also add a heading "Figure legends".
-I attach to this email a related ms file with comments by our data editors. Please address all comments in the final manuscript.
I would like to suggest a few minor changes to the abstract that needs to be written in present tense. I also modified a few sentences. Please check carefully and let me know whether anything is incorrect: Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C-terminal fragments of ~25 kDa ("TDP-25") accumulate in cytoplasmic inclusions. Here, we analyze gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics and functional assays. In neurons, cytoplasmic TDP-25 inclusions are amorphous, and photobleaching experiments reveal gel-like biophysical properties that are less dynamic than nuclear TDP-43 is. Compared with full length TDP-43, the TDP-25 interactome is depleted of low-complexity domain proteins. TDP-25 inclusions are enriched in 26S proteasomes in exclusively substrate-processing conformations, suggesting that inclusions sequester proteasomes that are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP-25 impairs proteostasis, and this inhibitory function is enhanced by ALS-causing TDP-25 [or TDP-43??] mutations. These findings support a patho-physiological relevance of proteasome dysfunction in ALS/FTD. I also slightly modified the short summary and bullet points. Do you agree with: TDP-25, a C-terminal fragment of TDP-43 found in ALS and FTD patients, forms cytoplasmic inclusions with gel-like properties in primary neurons. Proteasome enrichment and impaired proteostasis support a relevance of proteasome dysfunction in ALS/FTD. Authors have answered very well all the major issues raised by this reviewer Referee #3: The revised paper is much improved having clarified some and addition of stats has given more weight to the result. Overall, I would have liked to see some additional information as the response letter gives a nice amount of detail but I understand that the paper has length restrictions.
Referee 1's comment on referee 2's concerns: a) The first major concern of the reviewer was about the way aggregates were picked (basically a technical query). To this, the authors answered fully and better explained the procedure. b) The second query was not really a concern but just the need for a clarification regarding the localization of the aggregates (and authors complied) c) The third query was again just a curiosity: whether there were Cryo-ET differences between the WT and mutant proteins (there were not). Importantly, neither answer would have impacted the study or any further interpretation of the data. d) The fourth query was a potentially major concern because it was not clear to this reviewer what had been the methodology to pick ribosomes, proteasome and TRiC. In my opinion, authors have also replied well to this query mentioning the fact that they used the same methodology that had been optimized in their 2018 Cell paper. e) Queries 5 and 6 were also mostly technical, dealing with the need to provide additional CryoET statistics. I am not an expert here but to me the reply of the authors looks quite professional. f) finally, query 7 was just a request to submit the structures to a data repository (which the authors did) Overall, my feeling therefore is that the authors have complied with all requirements outlined by this reviewer.

28th Mar 2022 2nd Authors' Response to Reviewers
The authors have addressed all minor editorial requests. I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to our journal.
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